Use of Ribozymes in the Detection of Adventitious Agents

ABSTRACT

The present invention provides a method of detecting adventitious agents in a composition comprising a microorganism by using ribozyme-expressing indicator cells, as well as indicator cells useful in such detection.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationsSer. No. 60/360,730, filed Feb. 28, 2002; and Ser. No. 60/441,760, filedJan. 23, 2003. The entire disclosure of these prior applications ishereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a method of detecting adventitious agents in acomposition comprising a microorganism, as well as indicator cellsuseful in such detection.

REFERENCES

-   U.S. Pat. No. 6,307,041.-   U.S. Pat. No. 6,136,307.-   U.S. Pat. No. 5,631,359.-   U.S. Pat. No. 5,631,115.-   U.S. Pat. No. 5,254,678.-   U.S. Pat. No. 5,168,053.-   U.S. Pat. No. 4,987,071.-   WO 91/04319.-   WO 91/04324.-   WO 94/18992.-   WO 94/25627.-   WO 99/18799.-   WO 02/095042.-   European Application No. 89 117 424.-   Andreansky, S. A., et al., “The application of genetically    engineered herpes simplex viruses to the treatment of experimental    brain tumors”, Proc. Natl. Acad. Sci. 93(21):11313-11318 (1996).-   Bar-Eli, N., et al., “preferential cytotoxic effect of Newcastle    disease virus on lymphoma cells”, J. Cancer Res. Clin. Oncol. 122:    409-415 (1996).-   Bergmann, M., et al., “A genetically engineered influenza A virus    with ras-dependent oncolytic properties”, Cancer Res. 61:8188-8193    (2001).-   Bischoff J R. et al., “An Adenovirus Mutant that Replicates    Selectively in p53-Deficient Human Tumor”, Science 274(5286):373-6    (1996).-   Blagoslelonny, M. V., et al., “in vitro Evaluation of a    p53-Expressing Adenovirus as an Anti-Cancer Drug”, Int. J. Cancer    67(3):386-392 (1996).-   Chandron and Nibert, “Protease cleavage of reovirus capsid protein    mu1 and mu1C is blocked by alkyl sulfate detergents, yielding a new    type of infectious suhvirion particle”, J. of Virology 72(1):467-75    (1998).-   Chang et al., J. Virol. 69:6605-6608 (1995).-   Chang et al., Proc. Natl. Acad. Sci. 89:4825-4829 (1992).-   Chang et al., Virol. 194:537-547 (1993).-   Coffey, M. C., et al., “Reovirus therapy of tumors with activated    Ras pathway”, Science 282: 1332-1334 (1998).-   Duncan et al., “Conformational and functional analysis of the    C-terminal globular head of the reovirus cell attachment protein”,    Virology 182(2):810-9 (1991).-   Farassati, F., et al., “Oncogenes in Ras signalling pathway dictate    host-cell permissiveness to herpes simplex virus 1”, Nat. Cell Biol.    3(8):745-750 (2001).-   Fields, B. N. et al., Fundamental Virology (3rd Edition),    Lippincott-Raven (1996).-   Fueyo, J., et al., “A Mutant Oncolytic Adenovirus Targeting the Rb    Pathway Produces Anti-Glioma Effect in Vivo”, Oncogene 19(1):2-12    (2000).-   Heise, C. et al., “Replication-selective adenoviruses as oncolytic    agents”, J. Clin. Invest. 105(7):847-51 (2000).-   Kawagishi-Kobayashi, M. et al., Mol. Cell. Biol. 17:4146-4158    (1997).-   Mah et al., “The N-terminal quarter of reovirus cell attachment    protein sigma 1 possesses intrinsic virion-anchoring function”,    Virology 179(1):95-103 (1990).-   Ncmunaitis, J., Invest. New Drugs 17:375-386 (1999).-   Nibert, M. L., Schiff, L. A., and Fields, B. N., “Reoviruses and    their replication”, pages 1557-96 in Fundamental Virology (Fields et    al., 3rd Edition), Lippencott-Raven Press, 1996.-   Pastan and Gottesman, “Multidrug resistance”, Annu. Rev. Med. 42:    277-286 (1991).-   Pyle, A. M., “Ribozymes: a distinct class of metalloenzymes”,    Science 261, 709-714 (1.993).-   Reichard, K. W., et al., “Newcastle Disease Virus Selectively Kills    Human Tumor Cells”, J. of Surgical Research 52:448-453 (1992).-   Romano et al., Mol. Cell. Bio. 18(12):7304-7316 (1998).-   Sambrook and Russell, Molecular Cloning (3^(rd) Ed.), CSHL Press,    New York (2001).-   Shahi, S. et al., “Ribozymes that cleave reovirus genome segment Si    also protect cells from pathogenesis caused by reovirus infection”,    Proc. Natl. Acad. Sci. USA 98:4104-4106 (2001).-   Sharp et al., Virology 250:302-315 (1998).-   Stojdl, D. F., et al., “Exploiting Tumor-Specific Defects in the    Interferon Pathway with a Previously Unknown Oncolytic Virus”, Nat.    Med. 6(7):821-825 (2000).-   Strong, J. E. and P. W. Lee, “The v-erbV oncogene confers enhanced    cellular susceptibility to reovirus infection”, J. Viral. 70:    612-616 (1996).-   Strong, J. E., et al., “Evidence that the Epidermal Growth Factor    Receptor on Host Cells Confers Reovirus Infection Efficiency”,    Virology 197(1): 405-411 (1993).-   Turner and Duncan, “Site directed mutagenesis of the C-terminal    portion of reovirus protein sigma1: evidence for a    conformation-dependent receptor binding domain”, Virology    186(1):219-27 (1992).-   Yoon, S. S., et al., “An Oncolytic Herpes Simplex Virus Type I    Selectively Destroys Diffuse Liver Metastases from Colon Carcinoma”,    FASEB J. 14:301-311(2000).-   Zaug, A. J. and Cech, T. R., “The intervening sequence RNA of    Tetrahymena is an enzyme”, Science 231:470-475 (1986).-   Zorn, U. et al., “Induction of Cytokines and Cytotoxicity against    Tumor Cells by Newcastle Disease Virus”, Cancer Biotherapy    9(3):22-235 (1994).

All of the publications, patents and patent applications cited above orelsewhere in this application are herein incorporated by reference intheir entirety to the same extent as if the disclosure of eachindividual publication, patent application or patent was specificallyand individually indicated to be incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

In the field of medical or biological sciences, there has always beenthe demand to produce large quantities of microorganisms such asviruses, bacteria, yeasts, other fungi, parasites and prions. Theresulting microorganisms can be used to isolate and purify microbialproteins, generate vaccines, or provide infectious microorganisms forlaboratory or medical studies. Recently, the new development of virustherapy has further necessitated the need for efficient production ofinfectious viruses.

Reovirus therapy (U.S. Pat. No. 6,136,307) is an example of virustherapy. Reovirus is a double-stranded RNA virus with a segmentedgenome. The receptor for the mammalian reovirus is a ubiquitousmolecule, therefore reovirus is capable of binding to a multitude ofcells. However, most cells are not susceptible to reovirus infection andbinding of reovirus to its cellular receptor results in no viralreplication or virus particle production. This is probably the reasonwhy reovirus is not known to be associated with any particular disease.

It was discovered recently that cells transformed with the ras oncogenebecome susceptible to reovirus infection, while their untransformedcounterparts are not (Strong et al., 1998). For example, whenreovirus-resistant NIH 3T3 cells were transformed with activated Ras orSos, a protein which activates Ras, reovirus infection was enhanced.Similarly, mouse fibroblasts that are resistant to reovirus infectionbecame susceptible after transfection with the EGF receptor gene or thev-erbB oncogene, both of which activate the ras pathway (Strong et al.,1993; Strong et al., 1996). Thus, reovirus can selectively infect andkill cells with an activated ras pathway.

Ras pathway activation accounts for a large percentage of mammalianrumors. Activating mutations of the ras gene itself occur in about 30%of all human tumors (Bos, 1989), primarily in pancreatic (90%), sporadiccolorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia(30%). Activation of factors upstream or downstream of ras in the raspathway is also associated with tumors. For example, overexpression ofHER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is commonin breast cancer (25-30%), and overexpression of platelet-derived growthfactor (PDGF) receptor or EGF receptor is prevalent in gliomas andglioblastomas (40-50%). EGF receptor and PDGF receptor are both known toactivate ras upon binding to their respective ligand, and v-erbB encodesa constitutively activated receptor lacking the extracellular domain.Accordingly, reovirus therapy, which is highly selective forras-associated tumor cells, can be used to treat a vast variety oftumors.

Reovirus can be produced and purified in bulk preparations (U.S. PatentApplication Publication Number 2002/0037576 A1). To ensure that thereovirus preparation does not contain adventitious agents which mayresult in undesired side effects, the preparation is validated by usinga susceptible cell line and anti-reovirus antibodies. Thus, the cellline is exposed to either the virus preparation alone, or the viruspreparation that has been neutralized by a reovirus-specificneutralizing antibody. If the antibody neutralized virus preparation isstill pathogenic to the cell line, the virus preparation must contain anadventitious virus or other organism. The preparation is then discardedor further purified.

This validating protocol is expensive, as it requires large amounts ofhigh affinity, high titer antibodies to neutralize the virus. Thisproblem is further exacerbated now that we can produce reovirus veryefficiently, and the requirement for antibody is even higher. Therefore,a more cost effective approach is desirable.

SUMMARY OF THE INVENTION

The present invention provides a method of validating microbialpreparations using a ribozyme that is specific for the microorganismbeing prepared. For example, a plasmid encoding a ribozyme thatspecifically cleaves the genome of reovirus can be introduced into cellsthat are susceptible to reovirus infection. The transfected cells, byexpressing the ribozyme, are capable of inactivating reovirus and thuswill not be infected by the virus. The ribozyme-expressing cells arethen subjected to a reovirus preparation, and any pathogenic effectscaused by the reovirus preparation will indicate that an adventitiousagent is present in the reovirus preparation. Conversely, the absence ofany pathogenic effect validates the preparation as having no detectableadventitious agents.

This method is also applicable to viruses or other microorganisms havinga DNA genome. Since the DNA genome must be transcribed into RNA forsuccessful infection by the microorganism, a ribozyme specific for theRNA transcript will inhibit infection as well as replication of themicroorganism. Again, if the ribozyme-expressing cells show anypathogenic effects due to the microbial preparation, an adventitiousagent must be present in the preparation. Similarly, this method can beused to validate preparations of prions as well.

Accordingly, the present invention provides a method of detecting thepresence of an adventitious agent in a composition comprising areovirus, comprising:

(a) providing a population of indicator cells that expresses a ribozyme,wherein the ribozyme is capable of specifically cleaving the genome ofthe reovirus;

(b) contacting the indicator cells with the composition under conditionsthat allow for cleavage of the genome of the reovirus by the ribozyme;and

(c) determining the effect of the composition on the indicator cells,wherein any pathogenic effect indicates the presence of an adventitiousagent in the composition in addition to the reovirus.

The cells may express the ribozyme transiently or permanently.Preferably, the cells comprise a ribozyme-encoding gene that isintegrated into the genome of the cells. The ribozyme may cleave anypart of the reovirus genome that is important for replication orinfection by reovirus. For example, the ribozyme may cleave the S1segment of reovirus, such as Rz-553 or Rz-984.

This method can be used to detect adventitious agents in any reovirus.The reovirus is preferably a mammalian reovirus, more preferably aserotype 3 reovirus, and most preferably a Dearing strain reovirus. Thereovirus may be a recombinant reovirus. The recombinant reovirus may begenerated by co-infection of mammalian cells with different subtypes ofreovirus. The recombinant reovirus may be naturally-occurring ornon-naturally-occurring. The recombinant reovirus may be from two ormore strains of reovirus, particularly two or more strains of reovirusselected from the group consisting of strain Dearing, strain Abney,strain Jones, and strain Lang. The recombinant reovirus may also resultfrom reassortment of reoviruses from different serotypes, such asselected from the group consisting of serotype 1 reovirus, serotype 2reovirus and serotype 3 reovirus. The recombinant reovirus may comprisenaturally-occurring variant coat protein coding sequences or mutatedcoat protein coding sequences.

The present invention can be applied to compositions of anymicroorganism, including any virus. Accordingly, the present inventionprovides a method of detecting the presence of an adventitious agent ina composition comprising a virus wherein the virus contains an RNAgenome or utilizes an RNA transcript to replicate, comprising:

(a) providing a population of indicator cells that expresses a ribozyme,wherein the ribozyme is capable of specifically cleaving the RNA genomeor RNA transcript of the virus;

(b) contacting the indicator cells with the composition under conditionsthat allow for cleavage of the RNA genome or RNA transcript of the virusby the ribozyme; and

(c) determining the effect of the composition on the indicator cells,wherein any pathogenic effect indicates the presence of an adventitiousagent in the composition in addition to the virus.

The cells may express the ribozyme transiently or permanently.Preferably, the cells comprise a ribozyme-encoding gene that isintegrated into the genome of the cells.

The virus may be a DNA virus or RNA virus. Preferably, the virus is anoncolytic virus, which is capable of selectively replicating inneoplastic cells. Preferred oncolytic viruses include, but are notlimited to, modified adenovirus, modified HSV, modified vaccinia virus,modified parapoxvirus orf virus, modified influenza virus,p53-expressing viruses, the ONYX-015 virus, the Delta24 virus, andvesicular stomatitis virus.

Another aspect of the present invention provides a method of validatinga composition comprising a microorganism, comprising:

(a) providing a population of indicator cells that expresses a ribozyme,wherein the ribozyme is capable of specifically cleaving the RNA genomeor RNA transcript of the microorganism to inhibit replication orinfection of the microorganism;

(b) contacting the indicator cells with the composition under conditionsthat allow for cleavage of the RNA genome or RNA transcript of themicroorganism by the ribozyme; and

(c) determining the effect of the composition on the indicator cells,wherein the absence of any pathogenic effect validates the compositionas having no detectable adventitious agent.

The microorganism is preferably a virus, more preferably a virus capableof selectively replicating in neoplastic cells, and most preferably areovirus.

Another aspect of the present invention provides an indicator celluseful for detecting an adventitious agent in a composition ofmicroorganism wherein the indicator cell permanently expresses aribozyme that is capable of cleaving the genuine or RNA transcript ofthe microorganism. Preferably, a gene coding tor the ribozyme isintegrated into the genome of the indicator cell. The microorganism ispreferably a virus and more preferably a reovirus. The cell ispreferably derived from the human embryonic kidney 293 cells (HEK 293cells).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of detecting adventitious agentsin a preparation of a microorganism by using a ribozyme that is specificfor the microorganism. Thus, a cell population susceptible to themicroorganism is transfected by an expressing vector encoding theribozyme and exposed to the composition comprising the microorganism.Infection and/or replication of the microorganism in the cells areinhibited by the ribozyme. Therefore, the microorganism does not causeany pathogenic effect on the cells. As a result, any sign of pathogeniceffect is indicative of the presence of an adventitious agent in themicroorganism preparation.

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

Definitions

An “adventitious agent” is an agent that is not intended to be includedin a composition. Preferably, an adventitious agent is an infectiousagent, namely an agent capable of infecting a cell.

“Infecting” a cell refers to the act of entering into and replicating ina cell.

A “ribozyme” is an RNA molecule or RNA derivative that is capable ofcatalytically cleaving another RNA (the “target RNA”). The ribozymes ofthe present invention may have the characteristics of naturallyoccurring ribozymes. For example, the ribozymes isolated fromTetrahymena thermophila has an eight base pair active site whichhybridizes to a target RNA sequence before cleaving the target (see, forexample, Zaug and Cech, 1986). Free guanosine or guanosine derivativesis required for this reaction, and a guanosine is added to the 5′ end ofcleaved RNA. The ribozymes of the present invention may also besynthetic ribozymes, such as those described in U.S. Pat. No. 5,254,678.These synthetic ribozymes have separate hybridizing regions andcatalytic regions; therefore, the hybridizing regions can be designed torecognize any target sequences. In addition, the cleaved RNA is notmodified by these ribozymes.

An “indicator cell” is a cell that expresses a ribozyme, which ribozymeis capable of cleaving and inactivating a microorganism to be validated.The indicator cell, when not expressing the ribozyme, is susceptible toinfection of the microorganism.

A “pathogenic effect” is an adverse effect on the growth or maintenanceof a cell, particularly the effects associated with microbialinfections. Pathogenic effects include, but are not limited to,cytophathic effect (CPE), cell rupture, inhibition of growth, inhibitionof protein synthesis, and apoptosis.

“Cytopathic effect” is an observable change in cell structure.Cytopathic effect may vary with cell types and cause of death, and canbe determined according to established knowledge in the art. Forexample, some of the most common effects of viral infection aremorphological changes such as (a) cell rounding and detachment from thesubstrate; (b) cell lysis; (c) syncytium formation; and (d) inclusionbody formation. Cytopathic effect shown by reovirus-infected cells, forinstance, is indicated by the cells becoming swollen and granular inappearance and the cell clumps breaking up.

“Validating” a composition, as used herein, means proving that thecomposition does not contain an adventitious agent that is detectable bythe method employed.

“Reovirus” refers to any virus classified in the reovirus genus, whethernaturally occurring, modified or recombinant. Reoviruses are viruseswith a double-stranded, segmented RNA genome. The virions measure 60-80nm in diameter and possess two concentric capsid shells, each of whichis icosahedral. The genome consists of double-stranded RNA in 10-12discrete segments with a total genome size of 16-27 kbp. The individualRNA segments vary in size. Three distinct but related types of reovirushave been recovered from many species. All three types share a commoncomplement-fixing antigen.

The human reovirus consists of three serotypes: type 1 (strain Lang orT1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strainAbney, T3D). The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (see, for example,Fields, B. N. et al., 1996).

The reovirus may be naturally occurring or modified. The reovirus is“naturally-occurring” when it can be isolated from a source in natureand has not been intentionally modified by humans in the laboratory. Forexample, the reovirus can be from a “field source”, that is, from ahuman who has been infected with the reovirus.

The reovirus may be modified but still capable of lytically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may be coated in a liposome or micelle (Chandron andNibert, 1998). For example, the virion may be treated with chymotrypsinin the presence of micelle forming concentrations of alkyl sulfatedetergents to generate a new infectious subvirion particle.

The reovirus may be a recombinant reovirus resulting from therecombination/reassortment of genomic segments from two or moregenetically distinct reoviruses. Recombination/reassortment of reovirusgenomic segments may occur in nature following infection of a hostorganism with at least two genetically distinct reoviruses. Recombinantvirions can also be generated in cell culture, for example, byco-infection of permissive host cells with genetically distinctreoviruses (Nibert et al. 1995).

Accordingly, the invention contemplates the recombinant reovirusresulting from reassortment of genome segments from two or moregenetically distinct reoviruses, including but not limited to, humanreovirus, such as type 1 (e.g., strain Lang), type 2 (e.g., strainJones), and type 3 (e.g., strain Dearing or strain Abney), non-humanmammalian reoviruses, or avian reovirus. The invention furthercontemplates recombinant reoviruses resulting from reassortment ofgenome segments from two or more genetically distinct reoviruses whereinat least one parental virus is genetically engineered, comprises one ormore chemically synthesized genomic segment, has been treated withchemical or physical mutagens, or is itself the result of arecombination event. The invention further contemplates the recombinantreovirus that has undergone recombination in the presence of chemicalmutagens, including but not limited to dimethyl sulfate and ethidiumbromide, or physical mutagens, including but not limited to ultravioletlight and other forms of radiation.

The invention further contemplates recombinant reoviruses that comprisedeletions or duplications in one or more genome segments, that compriseadditional genetic information as a result of recombination with a hostcell genome, or that comprise synthetic genes.

The reovirus may be modified by incorporation of mutated coat proteins,such as for example σ1, into the virion outer capsid. The proteins maybe mutated by replacement, insertion or deletion. Replacement includesthe insertion of different amino acids in place of the native aminoacids. Insertions include the insertion of additional amino acidresidues into the protein at one or more locations. Deletions includedeletions of one or more amino acid residues in the protein. Suchmutations may be generated by methods known in the art. For example,oligonucleotide site directed mutagenesis of the gene encoding for oneof the coat proteins could result in the generation of the desiredmutant coat protein. Expression of the mutated protein in reovirusinfected mammalian cells in vitro such as COS1 cells will result in theincorporation of the mutated protein into the reovirus virion particle(Turner and Duncan, 1992; Duncan et al., 1991; Mah et al., 1990).

The reovirus is preferably a reovirus modified to reduce or eliminate animmune reaction to the reovirus. Such modified reovirus are termed“immunoprotected reovirus”. Such modifications could include packagingof the reovirus in a liposome, a micelle or other vehicle to mask thereovirus from the mammals immune system. Alternatively, the outer capsidof the reovirus virion particle may be removed since the proteinspresent in the outer capsid are the major determinant of the hosthumoral and cellular responses.

A “neoplastic cell”, also known as a “cell with a proliferativedisorder”, refers to a cell which proliferates at an abnormally highrate. A new growth comprising neoplastic cells is a neoplasm, also knownas a tumor. A neoplasm is an abnormal tissue growth, generally forming adistinct mass, that grows by cellular proliferation more rapidly thannormal tissue growth. A neoplasm may show partial or total lack ofstructural organization and functional coordination with normal tissue.As used herein, a neoplasm is intended to encompass hematopoietic tumorsas well as solid tumors.

A neoplasm may be benign (benign tumor) or malignant (malignant tumor orcancer). Malignant tumors can be broadly classified into three majortypes. Malignant neoplasms arising from epithelial structures are calledcarcinomas, malignant neoplasms that originate from connective tissuessuch as muscle, cartilage, fat or bone are called sarcomas and malignanttumors affecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. Other neoplasms include, but are notlimited to neurofibromatosis.

“Ras-activated neoplastic cells” or “ras-mediated neoplastic cells”refer to cells which proliferate at an abnormally high rate due to, atleast in part, activation of the ras pathway. The ras pathway may beactivated by way of ras gene structural mutation, elevated level of rasgene expression, elevated stability of the ras gene message, or anymutation or other mechanism which leads to the activation of ras or afactor or factors downstream or upstream from ras in the ras pathway,thereby increasing the ras pathway activity. For example, activation ofEGF receptor, PDGF receptor or sos results in activation of the raspathway. Ras-mediated neoplastic cells include, but are not limited to,ras-mediated cancer cells, which are cells proliferating in a malignantmanner due to activation of the ras pathway.

An “oncolytic” virus is a virus that is capable of selectively infectingand killing neoplastic cells. in particular, the oncolytic virus iscapable of selectively replicating in and lysing neoplastic cells.Examples of oncolytic viruses include, bat are not limited to, modifiedadenovirus, modified HSV, modified vaccinia virus, modified parapoxvirusorf virus, modified influenza virus, p53-expressing viruses, theONYX-015 virus, the Delta24 virus, vesicular stomatitis virus, theherpes simplex virus 1 mutant which is defective in hrR3, Newcastledisease virus, encephalitis virus, herpes zoster virus, hepatitis virus,influenza virus, varicella virus, and measles virus.

The term “attenuated adenovirus” or “modified adenovirus” means anadenovirus in which the gene product or products which prevents theactivation of PKR is lacking, inhibited or mutated such that PKRactivation is not blocked. Preferably, the VAI RNA's are nottranscribed. Such attenuated or modified adenovirus would not be able toreplicate in normal cells that do not have an activated ras pathway,however, it would be able to infect and replicate in cells having anactivated ras pathway.

The term “attenuated RSV” or “modified HSV” means a herpes simplex virus(HSV) in which the gene product or products that prevents the activationof PKR is lacking, inhibited or mutated such that PKR activation is notblocked. Preferably, the HSV gene _(γ1)34.5 is not transcribed. Suchattenuated or modified HSV would not be able to replicate in normalcells that do not have an activated ras pathway, however, it would beable to infect and replicate in cells having an activated ras pathway.

“Parapoxvirus orf virus” is a poxvirus. It is a virus that induces acutecutaneous lesions in different mammalian species, including humans.Parapoxvirus on virus naturally infects sheep, goats and humans throughbroken or damaged skin, replicates in regenerating epidermal cells andinduces pustular leasions that turn to scabs (Haig et al., 1998). Theterm “attenuated parapoxvirus orf virus” or “modified parapoxvirus onvirus” means a parapoxvirus on virus in which the gene product orproducts which prevents the activation of PKR is lacking, inhibited ormutated such that PKR activation is not blocked. Preferably, the geneOV20.0L is not transcribed. Such attenuated or modified parapoxvirus orfvirus would not be able to replicate in normal cells that do not have anactivated ras pathway, however, it would be able to infect and replicatein cells having an activated ras pathway.

The term “attenuated vaccinia virus” or “modified vaccinia virus” meansa vaccinia virus in which the gene product or products which preventsthe activation of PKR is lacking, inhibited or mutated such that PKRactivation is not blocked. Preferably, the E3L gene and/or the K3L geneis not transcribed. Such attenuated or modified vaccinia virus would notbe able to replicate in normal cells that do not have an activated raspathway, however, it would be able to infect and replicate in cellshaving an activated ras pathway.

The term “attenuated influenza virus” or “modified influenza virus”means an influenza virus in which the gene product or products whichprevents the activation of PKR is lacking, inhibited or mutated suchthat PKR activation is not blocked. Preferably, the NS1 gene is nottranscribed. Such attenuated or modified influenza virus would not beable to replicate in normal cells that do not have an activated raspathway, however, it would be able to infect and replicate in cellshaving an activated ras pathway.

Methods

The present invention provides a method of detecting adventitious agentsin a composition comprising microorganisms. An embodiment of the methodis demonstrated in Example 1. Thus, to determine if an adventitiousagent is present in a reovirus preparation, a plasmid that encodes aribozyme, Rz-553 (Shahi et al., 2001), is constructed. Rz-553 is a“hammerhead” ribozyme consisting of a catalytic region flanked by twoeight-nucleotide sequences that hybridize to the S1 segment of thereovirus genome. S1 codes for the protein σ1, which binds to thereovirus receptor on the cell. Therefore, cleavage of S1 RNA leads toreduced infectivity of any resultant virus. Also constructed is aplasmid encoding a mutant of Rz-553 in which a single nucleotide in thecatalytic region is mutated (G to U). The mutant is known to becompletely inactive.

The plasmids are introduced into COS-1 cells, and the transfected cellsare exposed to an aliquot of the reovirus preparation being tested.Mock-infected cells are used as a control. As expected, cells expressingthe mutant ribozyme had extensive CPE. However, cells expressing Rz-553also show moderate CPE when compared to the mock-infected cells.Therefore, the reovirus preparation contains a non-reovirus agent thatcaused the CPE on COS-1 cells.

Any ribozyme capable of cleaving the target RNA sequence of amicroorganism to inhibit replication or infection of the microorganismis useful in the present invention. The target sequence may be, forexample, RNAs encoding structural proteins (particularly outer coatproteins), proteins of the replication machinery, or proteins importantfor cellular entry, such as the receptor protein. Methods forconstructing sequence-specific ribozymes are well known in the art. Forexample, U.S. Pat. No. 5,254,678 describes the hammerhead ribozymes,which have a central catalytic region flanked by two hybridizationregions. Upon hybridizing to the pre-selected target sequence throughthe hybridization regions, the catalytic region forms a secondarystructure that facilitates cleavage, and cleaves the target sequence.Although this patent describes ribozymes in which at least onehybridization region has a minimum of nine hybridizing nucleotides, suchminimal length is not required in the present invention. In the presentinvention, each hybridization region may contain six, seven, eight ormore hybridizing nucleotides.

U.S. Pat. No. 6,307,041 describes derivatives of hammerhead ribozymes,including the circular, hairpin, circular/hairpin, lariat andhairpin-lariat forms of hammerhead ribozymes. These ribozymes haveincreased specific activity and different co-factor requirement, and mayalso be used in the present invention. Other examples include thehairpin ribozymes as described in, for example, U.S. Pat. Nos. 5,631,359and 5,631,115.

All these ribozymes contain at least one hybridization region and acatalytic region. The hybridization region is designed according to thetarget sequence. The catalytic region can be derived from, for example,a hammerhead ribozyme (U.S. Pat. No. 5,254,678), a hairpin ribozyme(European Application No. 89 117 424), a hepatitis delta ribozyme (WO91/04319 and WO 91/04324), an RNase P ribozyme (U.S. Pat. No.5,168,053), a group I intron (U.S. Pat. No. 4,987,071), or a group IIintron (Pyle, 1993).

It is also contemplated that more than one ribozyme can be combined inthe present invention. For example, multiple ribozymes recognizingdifferent regions of the same RNA can be combined. Alternatively andpreferably, ribozymes specific for different RNAs of the samemicroorganism can be used together to increase the efficiency ofinactivation of the microorganism. When multiple ribozymes are used,they can be encoded in the same expression vector or separately encoded.

The present invention may be used to detect the presence of adventitiousagents in any microbial preparation. It should be noted that whileribozymes cleave RNA only, the application of the present invention isnot limited to microorganisms with an RNA genome. Microorganisms with aDNA genome necessarily need to replicate through an RNA transcript,and/or synthesize proteins through an RNA transcript, as part of theinfection process. Even prions need an RNA transcript to replicate andinfect. Therefore, replication and infection of any microorganism can beinhibited by a ribozyme that specifically cleaves the microbial RNAtranscript/genome.

The microorganism of the present invention is preferably a virus andmore preferably an oncolytic virus. An oncolytic virus can selectivelyinfect and kill neoplastic cells, thus is useful in virus therapy. Inaddition to reovirus, these viruses include, but are not limited to,modified adenovirus, modified HSV, modified vaccinia virus, modifiedparapoxvirus orf virus, modified influenza virus, p53-expressingviruses, the ONYX-015 virus, the Delta24 virus, vesicular stomatitisvirus, the herpes simplex virus 1 mutant which is defective in hrR3,Newcastle disease virus, encephalitis virus, herpes zoster virus,hepatitis virus, influenza virus, varicella virus, and measles virus.

Adenovirus, HSV, vaccinia virus, and parapoxvirus on virus are viruseswhich have developed a mechanism to overcome the double stranded RNAkinase (PKR). Normally, when virus enters a cell, PKR is activated andblocks protein synthesis, and the virus can not replicate in this cell.However, adenovirus makes a large amount of a small RNA, VA1 RNA. VA1RNA has extensive secondary structures and hinds to PKR in competitionwith the double stranded RNA (dsRNA) which normally activates PKR. Sinceit requires a minimum length of dsRNA to activate PKR, VA1 RNA does notactivate PKR. Instead, it sequesters PKR by virtue of its large amount.Consequently, protein synthesis is not blocked and adenovirus canreplicate in the cell. It should be noted, however, that although theprotein synthesis machinery is not blocked, host cell protein synthesisis inhibited by the virus to facilitate viral protein synthesis.

Vaccinia virus encodes two gene products, K3L and E3L, whichdown-regulate PKR with different mechanisms. The K3L gene product haslimited homology with the N-terminal region of eIF-2α, the naturalsubstrate of PKR, and may act as a pseudosubstrate for PKR. The E3L geneproduct is a dsRNA-binding protein and apparently functions bysequestering activator dsRNAs.

Similarly, herpes simplex virus (HSV) gene _(γ1)34.5 encodes the geneproduct infected-cell protein 34.5 (ICP34.5) that can prevent theantiviral effects exerted by PKR. The parapoxvirus orf virus encodes thegene OV20.0L that is involved in blocking PKR activity. Thus, theseviruses can successfully infect cells without being inhibited by PKR.

In the modified adenovirus, modified HSV, modified vaccinia virus, ormodified parapoxvirus orf virus, the viral anti-PKR mechanism has beenmutated or otherwise inactivated. Therefore, these modified viruses arenot capable of replicating in normal cells which have normal PKRfunction. Ras-activated neoplastic cells, however, are not subject toprotein synthesis inhibition by PKR, because ras inactivates PKR. Thesecells are therefore susceptible to infection by the modified adenovirus,modified HSV, modified vaccinia virus, or modified parapoxvirus orfvirus.

The viruses can be modified or mutated according to the knownstructure-function relationship of the viral PKR inhibitors. Forexample, since the amino terminal region of E3 protein of the vacciniavirus interacts with the carboxy-terminal region domain of PKR, deletionor point mutation of this domain prevents anti-PKR function (Chang etal., 1992, 1993, 1995; Sharp et al., 1998; Romano et al., 1998). The K3Lgene of vaccinia virus encodes pK3, a pseudosubstrate of PKR. There is aloss-of-function mutation within K3L. By either truncating or by placingpoint mutations within the C-terminal portion of K3L protein, homologousto residues 79 to 83 in eIF-2α abolish PKR inhibitory activity(Kawagishi-Kobayashi et al., 1997).

The modified HSV include, but are limited to, R3616 (both copies of the_(γ1)34.5 gene have been deleted), R4009 (two stop codons have beeninserted in the _(γ1)34.5 gene), and G207 (mutated in the ribonucleotidereductase and the _(γ1)34.5 genes) (Andreansky et al., 1996). Thesemodified viruses have been used in brain tumor therapy, and it has beenrecently shown that R3616 preferentially infects ras-activated cells(Farassati et al., 2001).

Similarly, the delNS1 virus (Bergmann et al., 2001) is a geneticallyengineered influenza A virus that can selectively replicate inras-activated neoplastic cells. The NS1 protein of influenza virus is avirulence factor that overcomes the PKR-mediated antiviral response bythe host. NS1 is knocked out in the delNS1 virus, which fails to infectnormal cells, presumably due to PKR-mediated inhibition, but replicatessuccessfully in ras-activated neoplastic cells. Therefore, a modifiedinfluenza virus in which NS1 is modified or mutated, such as the delNS1virus, is also useful in the present invention.

Other oncolytic viruses include the viruses which selectively killneoplastic cells by carrying a tumor suppressor gene. For example, p53is a cellular tumor suppressor which inhibits uncontrolled proliferationof normal cells. However, approximate half of all tumors have afunctionally impaired p53 and proliferate in an uncontrolled manner.Therefore, a virus which expresses the wild type p53 gene canselectively kill the neoplastic cells which become neoplastic due toinactivation of the p53 gene product. Such a virus has been constructedand shown to induce apoptosis in cancer cells that express mutant p53(Blagosklonny et al., 1996).

Adenoviruses carrying the E2 gene have also been developed (WO02/095042). The E2 gene encodes the E2 protein, which inhibits oncogeneexpression and induces cellular senescence. Therefore, adenovirusesexpressing the E2 gene can be used in gene therapy, particularly forcancer patients in the terminal stage.

A similar approach involves viral inhibitors of minor suppressors. Forexample, certain adenovirus, SV40 and human papilloma virus includeproteins that inactivate p53, thereby allowing their own replication(Nemunaitis 1999). For adenovirus serotype 5, this protein is a 55 Kdprotein encoded by the E1B region. If the E1B region encoding this 55 kdprotein is deleted, as in the ONYX-015 virus (Bischoff et al, 1996;Heise et al., 2000; WO 94/18992), the 55 kd p53 inhibitor is no longerpresent. As a result, when ONYX-015 enters a normal cell, p53 functionsto suppress cell proliferation as well as viral replication, whichrelies on the cellular proliferative machinery. Therefore, ONYX-015 doesnot replicate in normal cells. On the other hand, in neoplastic cellswith disrupted p53 function, ONYX-015 can replicate and eventually causethe cell to die. Accordingly, this virus can be used to selectivelyinfect and kill p53-deficient neoplastic cells. A person of ordinaryskill in the art can also mutate and disrupt the p53 inhibitor gene inadenovirus 5 or other viruses according to established techniques.

Another example is the Delta24 virus which is a mutant adenoviruscarrying a 24 base pair deletion in the E1A region (Fueyo et al., 2000).This region is responsible for binding to the cellular tumor suppressorRb and inhibiting Rb function, thereby allowing the cellularproliferative machinery, and hence virus replication, to proceed in anuncontrolled fashion. Delta24 has a deletion in the Rb binding regionand does not bind to Rb. Therefore, replication of the mutant virus isinhibited by Rb in a normal cell. However, if Rb is inactivated and thecell becomes neoplastic, Delta24 is no longer inhibited. Instead, themutant virus replicates efficiently and lyses the Rb-deficient cell.

Yet other oncolytic viruses include the interferon sensitive viruses.Vesicular stomatitis virus (VSV) selectively kills neoplastic cells inthe presence of interferon. Interferons are circulating factors whichhind to cell surface receptors which ultimately lead to both anantiviral response and an induction of growth inhibitory and/orapoptotic signals in the target cells. Although interferons cantheoretically be used to inhibit proliferation of tumor cells, thisattempt has not been very successful because of tumor-specific mutationsof members of the interferon pathway.

However, by disrupting the interferon pathway to avoid growth inhibitionexerted by interferon, tumor cells may simultaneously compromise theiranti-viral response. Indeed, it has been shown that VSV, an enveloped,negative-sense RNA virus rapidly replicated in and killed a variety ofhuman tumor cell lines in the presence of interferon, while normal humanprimary cell cultures were apparently protected by interferon. Anintratumoral injection of VSV also reduced tumor burden of nude micebearing subcutaneous human melanoma xenografts (Stojdl et al., 2000).

Other interferon-sensitive viruses (WO 99/18799), namely viruses whichdo not replicate in a normal cell in the presence of interferons, can beidentified by growing a culture of normal cells, contacting the culturewith the virus of interest in the presence of varying concentrations ofinterferons, then determining the percentage of cell killing after aperiod of incubation. Preferably, less than 20% normal cells is killedand more preferably, less than 10% is killed.

It is also possible to take advantage of the fact that some neoplasticcells express high levels of an enzyme and construct a virus which isdependent on this enzyme. For example, ribonucleotide reductase isabundant in liver metastases but scarce in normal liver. Therefore, aherpes simplex virus 1 (HSV-1) mutant which is defective inribonucleotide reductase expression, hrR3, was shown to replicate incolon carcinoma cells but not normal liver cells (Yoon et al., 2000).

In addition to the viruses discussed above, a variety of other viruseshave been associated with tumor killing, although the underlyingmechanism is not always clear. Newcastle disease virus (NDV) replicatespreferentially in malignant cells, and the most commonly used strain is73-T (Reichard et al., 1992; Zorn et al, 1994; Bar-Eli et al, 1996).Clinical antitumor activities wherein NDV reduced tumor burden afterintratumor inoculation were also observed in a variety of tumors,including cervical, colorectal, pancreas, gastric, melanoma and renalcancer (WO 94/25627; Nemunaitis, 1999).

Moreover, encephalitis virus was shown to have an oncolytic effect in amouse sarcoma tumor, but attenuation may be required to reduce itsinfectivity in normal cells. Tumor regression have been described intumor patients infected with herpes zoster, hepatitis virus, influenza,varicella, or measles virus (for a review, see Nemunaitis, 1999). Theseviruses are thus also candidate oncolytic viruses.

it is contemplated that for the modified oncolytic viruses, in which anucleic acid is modified to result in replication in tumor cells, theribozyme can be designed to cleave the RNA that corresponds to themodified nucleic acid. The ribozyme can be introduced into thesusceptible tumor cells to prepare the indicator cells. Since theribozyme cleaves the modified RNA, which is responsible for infectivityin the tumor cells, no infection will occur unless there is anadventitious agent in the virus preparation.

Indicator Cells

The indicator cell should be prepared from a cell that is susceptible toinfection of the microorganism to be validated. A construct encoding theappropriate ribozyme can be introduced into the susceptible cell by anymethod known in the art, such as calcium phosphate precipitation,liposome fusion, lipofectin®, electroporation, and viral infection.

Both transient and stable introduction of the construct may be useful inthe present invention. However, the indicator cells preferably expressthe ribozyme in a permanent manner. To this end, an expression vectorencoding the ribozyme may be integrated into the genome of the cell orintroduced as an episomal vector (such as a bovine papilloma virusvector). Typically, the expression vector contains a selectable marker,cells harboring the expression vectors are selected using the selectablemarker and maintained under selection. Methods for constructing suchvectors, transfection and selection are well known in the art (see, forexample, Sambrook, 2001).

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Abbreviations not defined have their generally acceptedmeanings.

-   ° C.=degree Celsius-   hr=hour-   min=minute-   μM=micromolar-   mM=millimolar-   M=molar-   ml=milliliter-   μl=microliter-   mg=milligram-   μg=microgram-   FBS=fetal bovine serum-   PBS=phosphate buffered saline-   DMEM=Dulbecco's modified Eagle's medium-   α-MEM=α-modified Eagle's medium-   β-ME=β-mercaptoethanol-   MOI=multiplicity of infection-   PFU=plaque forming units-   PKR=double-stranded RNA activated protein kinase-   EGF=epidermal growth factor-   PDGF platelet derived growth factor-   CPE=cytopathic effect

Example 1 Testing a Reovirus Preparation

Reovirus is prepared and purified according to U.S. patent applicationSer. No. 09/920,012, Briefly, human embryonic kidney 293 SF (293/SF)cells are cultured in 15 L spinner flasks and infected with reovirus ata multiplicity of infection of 0.5 when cell density reach 10⁶/ml. Theculture is incubated until cell lysis begins, as evidenced by theculture media color change from red to orange due to the presence ofphenol red in the media, or by a viable cell count under the microscope.At this point, the cells are harvested by centrifugation, resuspendedand disrupted by freeze/thaw. The virus is then purified by a cesiumchloride gradient.

COS-1 cells are used as an indicator cell line. Thus, COS-1 cells aregrown to 60% confluency in 6-well plates and transfected with Rz-553encoding DNA or control DNA (mutant S1-Rz-553) by Using Lipofectin(GIBCO/BRL) as described in Shahi et al. (2001). pSVLacZ (Promega) isco-transfected in all the experiments to ensure uniform transfectionefficiency. 12 hours later, the cells are washed once with fresh mediumand infected with an aliquot of the reovirus preparation described aboveat an m.o.i. of 1 PFU/cell. Mock-infected cells are treated in the samemanner without the reovirus preparation. 8 hours after infection, thecells are observed for cytopathic effects (CPE).

The mutant S1-Rz-553 transfected cells show extensive CPE as expected,since the mutant ribozyme does not inactivate reovirus and reovirusinfection causes CPE on the infected cells. However, the Rz-553transfected cells also display detectable CPE as compared tomock-infected cells. Since Rz-553 is known to inactivate reovirus, theseresults indicate that this reovirus preparation contains a non-reovirusagent that causes cytopathic effects on COS-1 cells.

1-16. (canceled)
 17. A method of detecting the presence of anadventitious agent in a composition comprising a virus wherein the viruscontains an RNA genome or utilizes an RNA transcript to replicate,comprising: (a) providing a population of indicator cells that expressesa ribozyme, wherein the ribozyme is capable of specifically cleaving theRNA genome or RNA transcript of the virus to inhibit replication orinfection of the virus; (b) contacting the indicator cells with thecomposition under conditions that allow for cleavage of the RNA genomeor RNA transcript of the virus by the ribozyme; and (c) determining theeffect of the composition on the indicator cells, wherein any pathogeniceffect indicates the presence of an adventitious agent in thecomposition in addition to the virus.
 18. The method of claim 17 whereinthe indicator cells express the ribozyme from a gene that is integratedinto the genome of the cells.
 19. The method of claim 17 wherein thevirus is a DNA virus.
 20. The method of claim 17 wherein the virus iscapable of selectively infecting neoplastic cells.
 21. The method ofclaim 17 wherein the virus is selected from the group consisting ofmodified adenovirus, modified HSV, modified vaccinia virus, modifiedparapoxvirus orf virus, modified influenza virus, p53-expressingviruses, the ONYX-015 virus, the Delta24 virus, and vesicular stomatitisvirus.
 22. A method of validating a composition comprising amicroorganism, comprising: (a) providing a population of indicator cellsthat expresses a ribozyme, wherein the ribozyme is capable ofspecifically cleaving the RNA genome or RNA transcript of themicroorganism to inhibit replication or infection of the microorganism;(b) contacting the indicator cells with the composition under conditionsthat allow for cleavage of the RNA genome or RNA transcript of themicroorganism by the ribozyme; and (c) determining the effect of thecomposition on the indicator cells, wherein the absence of anypathogenic effect validates the composition as having no detectableadventitious agent.
 23. The method of claim 13 wherein the microorganismis a virus.
 24. The method of claim 13 wherein the microorganism is avirus capable of selectively infecting neoplastic cells.
 25. The methodof claim 13 wherein the microorganism is a reovirus.
 26. The method ofclaim 13 wherein the microorganism is selected from the group consistingof modified adenovirus, modified HSV, modified vaccinia virus, modifiedparapoxvirus orf virus, modified influenza virus, p53-expressingviruses, the ONYX-015 virus, the Delta24 virus, and vesicular stomatitisvirus.
 27. An indicator cell useful for detecting an adventitious agentin a composition of microorganism wherein the indicator cell permanentlyexpresses a ribozyme that is capable of cleaving the genome or RNAtranscript of the microorganism.
 28. The indicator cell of claim 27wherein a gene coding for the ribozyme is integrated into the genome ofthe cell.
 29. The indicator cell of claim 27 wherein the microorganismis a virus.
 30. The indicator cell of claim 27 wherein the microorganismis reovirus.
 31. The indicator cell of claim 27 wherein the cell isderived from human embryonic kidney 293 cells.
 32. The method of claim17, wherein the virus is an interferon sensitive virus.
 33. The methodof claim 17, wherein the virus is an immunoprotected reovirus.
 34. Themethod of claim 23, wherein the virus is an interferon sensitive virus.35. The method of claim 23, wherein the virus is an immunoprotectedreovirus.